Results:

Serum CETP and IGF-1 levels were lower, whereas AdipoQ concentrations were higher in P compared with FL1 and FL2 members (CETP: p = 0.03 for both comparisons; IGF-1 p < 0.001 for both comparisons and ADIPOQ: p = 0.001 and p = 0.004, respectively). Furthermore, serum triglycerides, UA and glucose concentrations were higher in FD1 compared with FD2 subjects (p=0.001, 0.02 and ≤0.001, respectively). In FD2 and FL2, CETP levels were lower in individuals with B2B2 compared with B1B1 genotype (p=0.007). Additionally, ACE concentrations were higher in individuals with DD compared with II genotype in both Families (p=0.001). After adjustment for age and gender, CETP levels were lower in P and FL2 individuals with B2B2 compared with the B1B1 genotype (p=0.004 and 0.007, respectively).

Conclusion:

Increase serum TGs, UA and GL concentrations were higher in the middle-aged individuals compared with their children in families independently of their lifespan. The serum adiponectin concentration was the highest in the oldest old individuals implying beneficial influence on lifespan. Independently of family’s lifespan history, the youngest individuals with CETPB2B2 genotype, compared with individuals with CETPB1B1 genotypes, had lower serum CETP concentrations. The knowledge of the unfavourable gene(s)influencing human lifespan may be helpful in encouraging individuals to follow healthier lifestyle habits and better control their high-risk biomarkers.

Unique Statistics:

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1. INTRODUCTION

Up-to-date, genetic studies have identified a limited number of loci associated with human longevity by recognizing age at death or survival up to advanced ages as a specific phenotype. Long-lived people are those who have exceeded ≥90 years [1Kolovou G, Barzilai N, Caruso C, et al. The challenges in moving from ageing to successful longevity. Curr Vasc Pharmacol 2014; 12(5): 662-73.[http://dx.doi.org/10.2174/1570161111666131219095114] [PMID: 24350930] ], some of them having overcome or stabilized or avoided deadly diseases such as cancer and atherosclerosis. The abovementioned characteristics of long-lived populations which led to their longer lifespan imply that beyond environmental factors, genes controlling lifespan may play an important role [2Kolovou GD, Kolovou V, Mavrogeni S. We are ageing. BioMed Res Int 2014; 2014: 808307.[http://dx.doi.org/10.1155/2014/808307] [PMID: 25045704] ].

In the present study, we evaluated serum levels of specific proteins as well as variants of their related genes in long-lived families comprising 3 generations and short-lived families comprising 2 generations. It is suggested that some people may have protective genes and proteins in contrast to people with a disease history “cargo”. This is the first time that potential longevity genes were studied in comparison with their serum protein levels in Greek families. A unique characteristic of this study was that samples were collected from all generations of both family groups, even from the oldest old members. The aim of this study was to investigate the differences in gene variants and serum protein levels between the members of long- and short-lived families.

2. SUBJECTS AND METHODS

2.1. Sample Collection

This study was designed and performed in agreement with the recommendations for the human genotype-phenotype association studies published by the National Cancer Institute-National Human Genome Research Institute (NCI-NHGRI) Working Group on Replication in Association Studies [33Chanock SJ, Manolio T, Boehnke M, et al. Replicating genotype-phenotype associations. Nature 2007; 447(7145): 655-60.[http://dx.doi.org/10.1038/447655a] [PMID: 17554299] ] indicating time and location of subject recruitment, success rate for DNA acquisition, internal control samples (from the same DNA) and sample tracking methods.

The study protocol was approved by the institutional ethics committee (Onassis Cardiac Surgery Center, Athens, Greece) and the Harokopio University (Athens, Greece) and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2000). All participants were of Caucasian origin and descent for ≥3 generations.

The Longevity Group (LON) consisted of the oldest old aged ≥90 years (P), one of their children (FL1) and one of their grandchildren (FL2). Families whose both parents died <75 years of any aged-related disease and had no history of individuals living >90 years [Early Death Group (EAD)] consisted of middle-aged individuals (FD1) and one of their children (FD2).

2.2. Genotyping

Genotyping was performed specifically for research purposes. Extraction of genomic DNA was performed from leukocytes separated from whole blood using a standard method with FlexiGene® DNA kit (Qiagen, Venlo, Netherlands).

The study variants were detected using Polymerase Chain Reaction (PCR) and Restricted Fragment Length Polymorphism analysis (RFLP) (details are included in the Supplementary Data).

RFLP results were validated in the following way: 1) around 20% of all samples were repeated to confirm findings of the PCR-RFLP method, and, 2) randomly selected PCR-RFLP results were confirmed by direct automated sequencing of PCR products for each polymorphism using the BigDye terminator chemistry kit (ABI, USA) and the 3,500 genetic analyser (ABI, USA). The concordance between repeated samples, sequencing and our results was 100%.

2.3. ELISA Measurements

Up to 4 ml of blood samples were obtained in BD (Becton Dickinson Diagnostics, NJ, USA) vacutainers. The blood was stored at room temperature for 45 min, after which the serum was separated by centrifugation at 1500xg, divided into aliquots, snap frozen and stored at -80°C until assay. The serum levels of each component were measured using commercially available Quantikine human ELISA kits: R&D systems, Minneapolis, MN, USA for AdipoQ, IGF-1, ACE and ALPCO Diagnostics, Salem, NH for CETP. The tests were performed according to the manufacturer’s specifications for each ELISA kit. The sensitivity of the assay for AdipoQ was 0.246 ng/mL, for IGF-1 0.026 ng/mL, for ACE 0.019 ng/mL and for CETP 0.2 μg/mL. The assay range for AdipoQ was 0.9-21.4 μg/mL, for IGF-1 40-258 ng/mL, for ACE 37.2-202 ng/mL and for CETP 0.2-5.0 μg/mL.

Body Mass Index (BMI) was calculated according to the following formula: weight (kg)/ [height (m)]2. In the P group, usually, the weight (kg) and height (m) were measured with difficulty due to posture problems. This was carried out at the patient’s home by a doctor who was taking the blood samples. In all other groups the weight and height were measured by a nurse in Outpatient Clinics.

2.5. Statistical Analysis

The normality of continuous variables was tested using the Shapiro-Wilk test. Levels of the quantitative variables are presented as median (25-75 percentile). Comparisons of mean values of age, BMI, TC, TGs, HDL-C, LDL-C UA, GL, CETP, ACE, IGF-1 and ADIPOQ between study groups (FD1-FD2 in the EAD Group, P-FL1, FL1-FL2, P-FL2 in the LON Group, and FL1-FD1, FL2-FD2 for LON vs EAD) were performed with the student unpaired t test. Categorical variables were compared with the use of the chi-square and Fisher’s exact tests. In order to assess the association of serum CETP, ACE, IGF1 and AdipoQ levels with the examined genotypes, multivariate regression analysis of gene polymorphisms and their related proteins, after adjustment for age and gender, was performed (p values of 0.05 were considered significant). The Bonferroni correction was used for the comparison (by one-way ANOVA) of mean values of TC, TGs, HDL-C, LDL-C, UA, GL, CETP, ACE, IGF-1 and ADIPOQ between EAD vs LON groups. Therefore, p value based on the Bonferroni correction is a=0.05/2 (number of statistical tests between 2 groups) x 10 (number of factors of interest) =0.0025.

3. RESULTS

Table 1Comparison of biochemical data between the Longevity and the Early Death Group.

The BMI among members of the LON Families should be interpreted with caution, since the height in P individuals was difficult to measure accurately. Serum TGs were lower in FL2 members compared with P and FL1, whereas serum HDL-C was higher in FL2 compared with FL1 members (p = 0.001 and p = 0.002, respectively). A trend towards higher serum UA levels in P compared with FL2 individuals was found (p = 0.06). Serum GL levels were higher in P compared with FL2 and in FL1 compared with FL2 (p = 0.002 and p = 0.04, respectively). Serum CETP and IGF-1 levels were lower, whereas AdipoQ concentrations were higher in P compared with FL1 and FL2 members (CETP: p = 0.03 for both comparisons; IGF-1 p < 0.001 for both comparisons and ADIPOQ: p = 0.001 and p = 0.004, respectively).

3.3. Comparison of Demographic and Clinical Data between the LON and EAD Groups

3.4. Relationships of Genotypes According to Encoding Protein

In the FD2 individuals of the EAD Group, serum CETP levels were lower for those with the B2B2 genotype (FD2: b = - 0.36, p = 0.05) as well as in the LON Group (FL2: b = - 0.45, p = 0.03), in comparison to the B1B1 genotype (Table 2a).

Table 2aAssociations between gene polymorphisms and their related proteins.

Table 2bMultivariate regression analysis of gene polymorphisms and their related proteins after adjustment for age and gender.

4. DISCUSSION

In the present study, in line with a previous one [34Kolovou V, Fragopoulou E, Antonopoulou S, Kolovou G. Influence of genes on the lifespan of long- and short-lived families. Hellenic J Cardiol 2017; 58(3): 228-32.[http://dx.doi.org/10.1016/j.hjc.2017.01.002] [PMID: 28081978] ], we compared biochemical and genetic markers between members of families with different lifespans.

The FL1 participants were 5 years older than FD1 group. There is no explanation for this and the difference is too small to have any influence on evaluated proteins levels as well as variants of their related genes.

In the LON Group, HDL-C was not significantly different in the FL2 compared with the FL1 group (the difference was only 1 mg/dl). In contrast, HDL-C was significantly higher in the FL2 compared with the P group, but the number of samples was small.

Independently of family’s lifespan history, the FD2 and FL2 individuals with CETPB2B2 genotype, compared with individuals with CETPB1B1 genotypes, had lower serum CETP levels (p = 0.05, p = 0.03, respectively). However, after adjustment for age and gender, only individuals from the LON group, and more specifically, P and FL2 individuals, had this association. This suggests the possibility of genetic determination in these families and that the B1 allele is unfavourable for long life. Furthermore, low CETP concentrations may act protectively in conjunction with the B2 allele.

It is well-established that the B2 homozygotes have less serum CETP activity or mass than B1 homozygotes (see review by Boekholdt and Thompson [12Boekholdt SM, Thompson JF. Natural genetic variation as a tool in understanding the role of CETP in lipid levels and disease. J Lipid Res 2003; 44(6): 1080-93.[http://dx.doi.org/10.1194/jlr.R200018-JLR200] [PMID: 12639975] ]). However, we found this correlation only in the families with longevity.

Human lifespan depends mainly on 2 factors: 1) expression of genes and epigenetics; both are responsible for plasma proteins, enzymes and molecule levels, and, 2) environmental effects, which can influence these variables. Therefore, we evaluated families with longevity or early deaths, so that all members of the same family had potentially similar lifestyle behaviour. The family environment has a critical role in the development of cardiometabolic disorders (such as smoking behaviour, eating habits, obesity and hypertension) in offspring and their children [64Ejtahed HS, Heshmat R, Motlagh ME, et al. Association of parental obesity with cardiometabolic risk factors in their children: The CASPIAN-V study. PLoS One 2018; 13(4): e0193978.[http://dx.doi.org/10.1371/journal.pone.0193978] [PMID: 29641604] ]. Additionally, the association of support from family for adoption of healthy eating habits and performing exercise with improvements of self-leadership (defined as a process of behavioural and cognitive self-evaluation and self-influence, in which an individual achieves the self-direction and self-motivation needed to make positive changes in behaviours) is also important as shown in cancer patients [65Lee MK, Park SY, Choi GS. Association of support from family and friends with self-leadership for making long-term lifestyle changes in patients with colorectal cancer. Eur J Cancer Care (Engl) 2018; 27(3): e12846.[http://dx.doi.org/10.1111/ecc.12846] [PMID: 29635763] ]. Thus, the parental lifespan history, biochemical phenotype and certain genes could be used as a practical approach for the early preventive measures and identification of families at risk for early death.

The main limitation of this study is the relatively small sample size. However, long-lived individuals represent a very selective group and it is therefore difficult to collect samples from these families. In the present study, samples were collected from all generations of both Family Groups, even from the oldest old members. Furthermore, it is the first time that Greek families both with a history of early death and with long lifespan were evaluated for potential longevity genes and biomarkers.

CONCLUSION

Increase serum TGs, UA and GL concentrations were higher in the middle-aged individuals compared with their children in families independently of their lifespan. Serum adiponectin concentration was the highest in the oldest old individuals implying beneficial influence on lifespan. Independently of family’s lifespan history, the youngest individuals with CETPB2B2 genotype, compared with individuals with CETPB1B1 genotypes, had lower serum CETP concentrations. The knowledge of the unfavourable gene(s) influencing human lifespan may be helpful in encouraging individuals to follow healthier lifestyle habits and better control their high-risk biomarkers.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The study protocol was approved by the institutional ethics committee (Onassis Cardiac Surgery Center, Athens,
Greece) and the Harokopio University (Athens, Greece).

HUMAN AND ANIMAL RIGHTS

No animals were used in this research. All research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2008 (http://www.wma.net/en/20activities/10ethics/10helsinki/)

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

ACKNOWLEDGEMENTS

Declared none.

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publishers Web site along with the published article.